Evaluating the performance of an ETD-Cleavable Cross-Linking strategy for Elucidating Protein Structures

Abstract

Chemical cross-linking is a powerful strategy for elucidating the structures of protein or protein complexes. The distance constraints obtained from cross-linked peptides represent the three-dimensional structures of the protein complexes. Unfortunately, structural analysis using cross-linking approach demands a significant amount of data to elucidate protein structures. This requires the development of several cleavable cross-linkers with different range of spacer chains. An Electron Transfer Dissociation (ETD) tandem mass spectrometry cleavable bond hydrazone was reported. Its fragmentation with conjugated peptides showed promise for the development of a new ETD cleavable cross-linker. However, no cross-linker was developed utilizing this ETD cleavable bond. For the first time, we attempted to develop an ETD cleavable cross-linker utilizing a hydrazone bond. We overcome the pitfall for the synthesis of this cross-linker and an easy synthesis scheme is reported. In this report, we evaluated the performance of this cross-linker called Hydrazone Incorporated ETD cleavable cross-linker (HI-ETD-XL) in model peptides and proteins. The characteristic fragmentation behavior of HI-ETD-XL during electron transfer dissociation and subsequent sequence identification of the peptide fragment ions by tandem mass spectrometry allowed the identification of cross-linked peptides unambiguously. We believe the availability of this ETD cleavable cross-linker will advance structural proteomics research significantly.

Introduction:

Chemical cross-linking coupled with mass spectrometric analysis (XL-MS) is becoming an alternative method for protein structural studies. This strategy has some advantages over the traditional approaches to characterize the structure of a protein or large protein complexes.[1, 2]

The core of the XL-MS analysis is a cross-linking reagent that covalently captures two proximal amino acid residues of protein or protein complexes. The cross-linker is composed of two reactive groups separated by a defined spacer length, which specifies the spatial orientation and distance information of the interacting proteins in multiprotein assembly. Usually, cross-linked products are identified using the bottom-up approach (LC-MS/MS), which utilizes enzymatic digestion to produce smaller peptides. The site of cross-linked amino acids along with the distance-constraint information, predicts a three-dimensional structural model of protein complexes.[3–7]

Due to the challenge of identifying cross-linked peptide with high confidence, a cleavable cross-linking approach was developed. These reagents contain one or more than one labile bond in their structures and can be preferentially cleaved in different ways based on their chemical properties. For example, cleavage of the labile bonds can be induced by photo, chemical, and gas-phase collision energy, which separates two inter cross-linked peptides before or during the MS analysis.[4, 8–12] Gas-phase MS-cleavable approach is most attractive due to its unique power to produce two diagnostic cross-link fragments in MS2 and subsequent identification of the cross-linked peptides accurately by MS3rd sequencing. [4, 13] Currently, most of the MS-cleavable reagents are collision-induced dissociation (CID)-cleavable, e.g. protein interaction reporters (PIR), disuccinimidyl sulfoxide (DSSO) and its derivatives, BuUrBu (DSBU), disuccinimidyl-succinamyl-aspartyl-proline (SuDP) and cyanurbiotindipropionylsuccinimide (CBDP).[9–11, 13–17] Recently, our group developed a novel Dual Cleavable Cross-linking Technology (DUCCT), which delivered high confidence identification of the same cross-linked peptides by differential tandem mass spectrometry. DUCCT conjugated cross-linked peptides cleaved by two tandem mass spectrometry fragmentation processes (CID and ETD) and generate two different signature ion peaks from the same cross-linked peptide. We proved that this dual cleavage workflow identified cross-linked peptides confidently.[4, 18]

Although Electron transfer dissociation (ETD) is a widely used tandem mass spectrometry fragmentation techniques, however, considerably less attention was paid to design ETD-cleavable cross-linkers compared to CID. ETD is a radical driven process that keeps many CID labile bonds un-cleaved, which is very suitable to keep the modification on amino acid side chains during fragmentation analysis. [19–21]. This feature makes ETD a reliable tool for characterizing the post-translation modifications (PTMs), e.g., phosphorylation, glycosylation.[22, 23]

The ETD appears to have a significant advantage in analyzing large, highly charged cross-linked peptides compared to collision-induced dissociation (CID). This process leads to comprehensive, uniformly distributed dissociation along peptide backbones, showing less impact on the peptide sequence than CID. Recently, the Heck Group has reported that ETD with the supplementary HCD (EThcD) activation generates the best sequence coverage for highly charged cross-linked species.[24, 25] One of the major concerns is the ETD fragmentation significantly depends on the precursor ion charge states. It is very crucial to increase the charge density of the selected precursor ions before activation. The charge density of the ions can be manipulated by covalent attachment of fixed charge sites and thus improve the fragmentation.[26]

The first ETD-cleavable cross-linker, DEB (1,3-diformyl-5-ethynylbenzene) was developed by the Burlingame group. DEB covalently links two interacting proteins or peptides through reductive animation and adds two protonation sites to the cross-linked moiety. [27, 28] Recently developed ETD-cleavable cross-linker, diethyl suberthioimidate (DEST), breaks at the amidino groups generated after reaction with primary amines. DEST cross-linked peptides identified as diagnostic ion pairs, consisting of peptide- NH₂ and peptide+linker+NH₃ ions in ETD-MS/MS. However, DEST hydrolyzed very quickly and generated high abundant hydrolyzed dead-end spectra that limit its application as well.[29, 30]

The mechanism of selective cleavage of the hydrazone bond was proposed by Jennifer Brodbelt group. First, they modified the peptides either with Succinimidyl 4-formylbenzoate (SFB) or succinimidyl 4-hydrazinonicotinate acetone hydrazone (SANH) and then the resultant peptides were conjugated together to form bis-arylhydrazone (BAH)-cross-linked peptides.[31–33] The selective breakdown of the hydrazone bond was observed under ETD fragmentation. This functional group improved overall charge density of the cross-linked peptides, and selective cleavage facilitated to generate the diagnostic ions in ETD-MS2 spectra. Further, CID-MS3 experiment confirmed the sequence of the peptides. Unfortunately, other than some fragmentation analysis no cross-linker was developed with hydrazone incorporated in the design. Additionally, their effectiveness in protein labeling was never demonstrated.

Structural analysis of an unknown protein by cross-linking is a challenging task. It requires significant experimental data from the treatment of several cross-linkers with different spacer chain lengths. Cross-linkers with different spacer chains and properties will produce volumes of confident experimental data, so that modeling study can be done effectively. Here, we introduced and evaluated a Hydrazone Incorporated ETD cleavable cross-linker (HI-ETD-XL), which will be very suitable for validation and benchmarking of protein structures. The cross-linker contains two reactive NHS ester groups for coupling lysine residues. We also incorporated a fixed-charge containing hydrazone bond, which can be preferentially cleaved using ETD-MS/MS. HI-ETD-XL protein labeling was demonstrated in different model peptides and proteins. We believe this HI-ETD-XL approach will be an additional structural tool kit for robust and quick structural analysis of proteins and protein complexes.

Materials and Methods:

Materials:

Two standard peptides, Neurotensin (pE-LYNKPRRPYIL) and Bradykinin (RPPGFSPFR) were ordered from Ana spec (San Jose, CA). Three proteins, Ubiquitin bovine erythrocytes, myoglobin of equine heart, and Bovine Serum Albumin (BSA) were obtained from Sigma-Aldrich (St. Louis, MO). The reducing agent, Dithiothreitol (DTT), was purchased from Bio-Rad (CA). Dimethyl sulfoxide (DMSO), Tris.HCl, Ammonium bicarbonate (ABC), and Formic acid (FA) were also obtained from the Sigma-Aldrich (St. Louis, MO). For proteolysis, sequencing-grade-modified trypsin ordered from Promega (Madison, WI, USA). Three killo Daltons (3K) MWCO protein concentrators (Pierce, IL) were utilized to remove excess cross-linking reagents and concentrate the protein after the cross-linking reaction. Pierce C18 Tip from Thermo Fisher Scientific (Rockford, IL, USA) was used to desalt the samples. Milli-Q-filtrated water (18 MΩ) (Aries Filter works, NJ) was used for all experimental studies.

The cross-linking reagent (HI-ETD-XL) was synthesized in our laboratory. Starting materials, 6-hydrazinicotinic acid purchased from AK Scientific (Union City, CA) and, EDC from Chem Impex (Wood Dale, IL). 4-formyl benzoic acid and N-hydroxysuccinimide (NHS) purchased from Sigma Aldrich. SiliaFlash® P60 40–63 μm (230–400 mesh) 60 Å Irregular Silica Gel used for flash column chromatography.

Synthesis of Cross-linking Reagent:

Detailed synthesis protocol was explained in the supplementary sections with proper characterization. Briefly, 4-formyl benzoic acid coupled with N-hydroxysuccinimide (NHS) (1.1 equiv.) in the presence of EDC and 2,5-dioxopyrrolidin-1-yl 4-formybenzoate formed as a white crystalline solid. In the second step, 2,5-dioxopyrrolidin-1-yl 6-(2-(propan-2-ylidene) hydrazinyl) nicotinate was synthesized after the coupling reaction of 6-hydrazinicotinic acid with N-hydroxysuccinimide (NHS) in the presence of triethylamine, acetone, and EDC. These two intermediate compounds then purified through silica-based column chromatography.

In final step, 2,5-dioxopyrrolidin-1-yl 4-formylbenzoate reacts with 2,5-dioxopyrrolidin-1-yl 6-(2-(propan-2-ylidene)hydrazinyl)nicotinate in presence of glacial acetic acid, resulting in a bright yellow solution of final pure product 2,5-dioxopyrrolidin-1-yl (E)-4-(2-(4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)benzylidene)hydrazinyl)-benzoate. Complete characterization and the NMR of the product was provided in the supplementary texts.

Cross-Linking Experiments:

Cross-Linking of standard Peptides:

The 100 mM stock solution of HI-ETD-XL cross-linker prepared in DMSO. Standard peptides (Neurotensin/Bradykinin) reacted with HI-ETD-XL in a 1:10 molar ratio in PBS buffer (pH 7.4). The cross-linking reaction was allowed to proceed for 30 mins at room temperature. Then, the reaction stopped by adding 50 mM Tris-HCl buffer (pH 8.5). After that, the samples were concentrated using a speed vacuum and desalted utilizing Pierce C18 zip tips. Desalted samples were dried and re-constituted with 49% Methanol/49% water/ 2% acetic acid (direct infusion solvent).

Protein Cross-Linking Experiments:

The efficiency of the cross-linking reagent tested with three different proteins, Ubiquitin, Myoglobin, and BSA. 1 mM of standard protein was prepared and reacted with the cross-linking reagent in a different molar ratio (1:50, 1:100, or 1: 200) in PBS buffer (pH 7.4). The reaction performed at room temperature in thermomixer for 30 mins. After 30 mins, 50 mM Tris.HCL Buffer was utilized to quench the reaction. 3K MWCO protein concentrators were used to remove the hydrolyzed cross-linking reagent and exchange the buffer with 50 mM ammonium bicarbonate buffer (pH 8). The concentration of the protein was determined with the BCA protein assay before digestion.

Digestion of Cross-linked Proteins:

Initially, the cross-linked proteins were reduced with 10 mM DDT and alkylated with 55 mM of iodoacetamide. Then sequence grade modified trypsin was added at a 1:50 (w/w) ratio of trypsin to protein in 50 mM ammonium bicarbonate buffer. Enzymatic digestion was allowed to continue for 16 h at 37 °C with 500 rpm constant rotation. Then the digested samples were concentrated by a speed vacuum, desalted using ZipTip, and re-constituted in 0.1% FA solution in water and stored at −20 °C.[34–36]

Nano HPLC/ Nano-ESI-LTQ Analysis:

Tryptic digests analyzed with LTQ Velos pro mass spectrometry combined with a UHPLC (UltiMate 3000, Dionex, USA). The digested peptides separated by a nano viper analytical C18 column. (Acclaim pepMap, 150 mm × 75 μm, 2 μm, 100 Å, Thermo Scientific, CA, USA). A 60 minutes gradient method performed to separate the digested peptides (0–3 min 4.0%B, 3–50 min 4.0–50.0% B, 50–50.1 min 50–90% B, 50.1–55 min 90% B, 55–55.1 min 90–4% B, 55.1–60 min 4% B; mobile phase A: 0.1% FA in water; mobile phase B: 0.1% FA in 95% acetonitrile, 5% water). 5 μl (partial injection mode) of digested samples injected at 300 nL/min flow.

The nano-electrospray ionization (ESI) source used with a fixed spray voltage at 2.4 kV and a heated capillary temperature at 275 °C. A full MS spectrum obtained in normal scan mode from 350 to 2000 m/z mass range. The data-dependent acquisition was performed using electron transfer based dissociation to get the MS/MS spectra for the 5 most abundant ions. For ETD mode, reagent source temperature, reagent ion source CI pressure, emission current, and reagent ion-electron energy set to 110°C, 20 psi, 50 UA, and −70 v, respectively. The ETD reaction time maintained at 100 ms, with an isolation width of 2 Da. The charge state of the ions filtered to >2 for ETD-MS/MS mode.

Data Analysis:

Data analysis of LC-MSⁿ spectra was carried out with our in-house developed software tool CLEAVE-XL. The detailed workflow of the Java coded CLEAVE-XL was provided in a recently published article.[18] The software is available to the scientific community upon request. Our specially designed software can identify the cross-linked peptides automatically from the dual cleavable (DUCCT/PC-DUCCT-Biotin) as well as single CID/ETD-cleavable cross-linkers. In the beginning, theoretical database of the peptides were generated based on the setting of proteolysis and amino acid sequences provided by the input protein fasta file. Next step, we created a cross-linked peptide database along with the fragment masses. This step was accomplished by adding the cross-linker mass (HI-ETD-XL) as well as fragment residual masses with the generated peptide masses. The experimental ETD-MS/MS data was processed as a MGF (Mascot Generic Format) file. Then, ETD-MS/MS experimental data file (MGF file) was searched against the theoretical database. A list of cross-linked peptides along with the reduced charged precursor-mass was generated in the ETD-output files. To reduce false-positive identification, intensity threshold of the MS/MS fragment peaks were set to 30% to filter the unambiguous ID.

Result and discussions:

Design and Synthesis of a Hydrazone Incorporated-ETD-Cleavable Cross-Linker (HI-ETD-XL).

In this study, we demonstrated a reasonably compact ETD-cleavable cross-linker (spacer chain length, ~14 Å) with different model peptides and proteins. The design of this cross-linker is shown in Figure 1A. This NHS containing cross-linker reacts with the primary amine group of the N-terminal or lysine residues. It contains one gas phase cleavable (N-N) bond, which preferentially cleaved by electron transfer-based dissociation before peptide backbone breakage. Selective cleavage of the Hydrazone Bond (N-N) by ETD was first reported by Jennifer Brodbelt group. Detail mechanism of the cleavage by ETD was described by Gardner et al. [31] Inspired by this study, we incorporated a hydrazine bond (N-N) bond in between two reactive NHS ester groups and synthesized this new gas-phase ETD-cleavable cross-linker, called HI-ETD-XL (Figure 1B). HI-ETD-XL was synthesized in the solution phase synthesis process, as demonstrated in the supplementary section. The spacer length distance is quite reasonable (~14 Å) for this ETD cleavable cross-linker, making it suitable for interactome study as well as structural studies of protein or protein complexes (Figure 1C). Considering the spacer length of this cross-linker and structural dynamics of the protein complexes, we estimated that a distance up to 35Å would be labeled.

Fig 1:

The design and chemical structure of the Hydrazone-Incorporated ETD Cleavable cross-linker (HI-ETD-XL). (A) The design of the ETD cleavable cross-linker. (B) The chemical structure of ETD MS-cleavable cross-linker. (C) The structure of HI-ETD-XL is shown with spacer chain length (~14 Å).

Cross-Linked Peptide Identification Strategies

Similar to other cleavable cross-linkers, HI-ETD-XL cross-linking can produce two types of cross-linked products: dead-end and inter cross-linked modified peptides. Inter cross-linked modified peptides provide essential information regarding spatial orientation and the distance constraints between the interacting residues. The protein identification workflow of the cross-linked peptides is illustrated in Figure 2/Figure S1. The cross-linker covalently connects the proteins with its interacting partner. Since HI-ETD-XL is an NHS-based cross-linker, it reacts with the nearby lysine residues of the protein. After tryptic digestion, cross-linked peptides are generated along with unmodified peptides. Finally, the cross-linked peptides are cleaved by ETD-MS/MS and further identified by CID-MS/MS tandem mass spectrometry. ETD preferentially cleaves the N-N bond (hydrazone) of the HI-ETD-XL cross-linked peptide and will generate two peptide peaks modified with cross-linker residual masses. Additionally, the generated charge-reduced ions will provide the charge states information of the precursor m/z if a low-resolution mass spectrometer is used. Dead-end peptides usually produce one diagnostic ion peak in ETD-MS/MS, and further MS3rd experiment will confirm the sequence of the fragment ions (Figure S1).

Fig 2:

The workflow for cross-linking proteins is shown with an inter cross-linked peptide. ETD cleavable cross-linker (HI-ETD-XL) is based on the hydrazone functional group. Preferential ETD cleavage site is pointed with a blue arrow. ETD-MS/MS leads to the selective cleavage on the N-N bond of hydrazone.

Characterization of HI-ETD-XL Cross-Linked Model Peptides by MSⁿ Analysis.

Initially, our synthesized cross-linker was characterized by NMR and Mass spectrum. The detailed description of the NMR spectra and Mass Spectrum was provided in the Supporting Information. ¹H & ¹³C NMR Spectra confirmed the structure of the cross-linker. Additionally, Full MS spectra provided the molecular weight (479.1077 Da) confirmation as well (Figure S2).

In Proof-of-Concept Studies, we tested the efficiency of cross-linker with two model peptides, namely Bradykinin (RPPGFSPFR) and Neurotensin (pELYENKPRR). Bradykinin has a free N-terminal in its sequence but doesn’t contain any lysine residue. On the other hand, Neurotensin has one lysine in its sequence, but its N terminal is blocked by a pyroglutamate group. We found that HI-ETD-XL efficiently reacted with both peptides and mostly formed inter-cross-linking products. The five charged-states precursor of an inter cross-linked peptide derived from the Bradykinin was identified at m/z 474.63. After ETD-MS/MS, a selective cleavage was observed at N-N bond, and two diagnostic fragment peaks were appeared at m/z 596.7 (2+) and 590.28 (2+). We also found the singly charge state peak of these modified peptides (m/z at 1180.65 and 1192.74) in the spectrum (Figure 3). Complete calculations of the cross-linked precursor and the fragment ion masses are provided in Table S1. A similar fragmentation pattern was observed for Neurotensin dimer. Signature fragment ions pinpointed the inter cross-linked dimer of the Neurotensin peptide (Figure S3).

Fig 3:

ETD-MS/MS of Bradykinin (RPPGFSPFR) dimer cross-linked with HI-ETD-XL (m/z 474.63, 5+). Selective cleavage was observed at N-N bond and generated two doubly charged diagnostic ions at m/z 590.28 and 596.37.

Characterization of HI-ETD-XL Cross-Linked Model Proteins by MSⁿ Analysis.

To verify the efficiency of the HI-ETD-XL for protein cross-linking in vitro, we used three different model proteins for our experiment, namely bovine serum albumin (BSA), bovine ubiquitin, and equine heart myoglobin. In-solution digestion of the cross-linked proteins was performed and resulted cross-linked peptides were subjected to LC–MSⁿ analysis.

MSⁿ analysis of a representative inter-cross-linked Myoglobin peptide, IPIKYLEFISDAIIHVLHSK-----LFTGHPETLEKFDK (m/z 850.66, 5+) displayed in Figure 4. The ETD-MS/MS spectrum generated a pair of diagnostic ions after preferential cleavage of the hydrazone bond that revealed the confident identification of the cross-linked peptide. Further, MS/MS of the fragment ions at m/z at 897.22 (2+) (LFTGHPETLEK*FDK) confirmed the sequence unambiguously (Figure 4B). Finally, MS2 diagnostic ions with MS3 sequencing confidently identified HI-ETD-XL inter cross-linked peptide (IPIKYLEFISDAIIHVLHSK…… LFTGHPETLEKFDK). Several dead-end peptides were identified efficiently from myoglobin. An ETD MS2 spectrum of HI-ETD-XL dead-end from myoglobin is displayed in Figure S4B. The triply charged precursor ion at 591.51 m/z confidently identified as HGTVVLTALGGILKK with a hydrolyzed dead end.

Fig 4:

MSⁿ Analysis of identified inter cross-linked peptides derived from Myoglobin protein. (A) ETD-MS/MS of cross-linked peptide IPIKYLEFISDAIIHVLHSK-----LFTGHPETLEKFDK (m/z 850.66, 5+). (B) CID-MS³ of one diagnostic fragment ion derived from ETD product ions.

After validation in a small protein, we evaluated the efficiency of HI-ETD-XL in a large protein, BSA (~66 KDa). A total of nine inter cross-linked peptides was identified from BSA after HI-ETD-XL cross-linking experiment (Table S2). An example of four-charge state BSA cross-linked peptide (ALKAWSVAR--------LAKEYEATLEECCAK) is provided in Figure 5. After applying electron transfer based energy, the hydrazone bond was cleaved preferentially and produced two signature ions, [(m/z = 973.34 (2+) and m/z = 1121.65 (1+)]. Further CID-MS3 experiment was performed to confirm the cross-linked peptide sequence. One of the MS/MS of the signature fragment peaks (LAKEYEATLEECCAK, 2+) derived from the inter peptide cross-linking is presented in Figure 5B.

Fig 5:

MSⁿ Analysis of one identified inter cross-linked peptide derived from BSA protein. (A) Characteristic fragmentation pattern of HI-ETD-XL cross-linked peptide (ALKAWSVAR--------LAKEYEATLEECCAK) derived from BSA. Two signature ions (m/z 973.34 and m/z 1121.65) were produced by ETD-MS/MS. (B) MS3 spectrum of one MS2 fragment ion (m/z 973.34) was demonstrated.

Again, an example of an identified dead-end peptide derived from BSA was displayed in Figure S4A. Highly abundant diagnostic ion peaks along with the reduced charged precursor ions pinpointed the triply charged dead-end peptide K*QTALVELLK unambiguously. We know that complete fragmentation of the precursor ions does not occur in ETD-MS/MS. However, we observed that highly charged cross-linked peptides derived from the HI-ETD-XL cross-linking showed efficient dissociation and highly abundant signature mass pairs. Sometimes, precursor ion and its charge-reduced ion masses were predominant in the MS2 spectra. Charge-reduced precursor ion is an excellent indicator of the charge states of the precursor ions. In Figure 5A, the HI-ETD-XL cross-linked peptide (ALKAWSVAR--------LAKEYEATLEECCAK) from BSA displayed diagnostic ion peaks and the reduced charge precursors with high intensity. A list of calculated masses and their m/z values used to assign cross-linked peptides derived from Myoglobin and BSA were provided in Table S3 and Table S4, respectively.

Figure 6 presents an example of a five-charge state inter cross-linked peptide (m/z at 781.56) identified from bovine ubiquitin. Two highly abundant characteristic fragment pairs, IQDK*EGIPPDQQR (m/z at 1131.09, 2+) and TLSDYNIQK*ESTLHLVLR (m/z at 822.61, 2+), were generated from the ETD-MS/MS is displayed in Figure 6. The high intensities of these diagnostic peaks confirmed that ETD favored N-N (Hydrazone) cleavage. MS3 analysis of diagnostic ions confirmed its sequence as IQDK*EGIPPDQQR and TLSDYNIQK*ESTLHLVLR, respectively (Figure S5A and S5B). Three inter cross-linked peptides were identified confidently using HI-ETD-XL from Ubiquitin. The list of inter-cross-linked peptides is shown in Table S5. The calculated masses of cross-linked precursors and fragment ions are provided in the Supplementary Information (Table S6).

Fig 6:

Example of one inter cross-linked peptide identified from Ubiquitin. ETD-MS/MS of cross-linked peptide (IQDKEGIPPDQQR--------TLSDYNIQKESTLHLVLR) (m/z 781.56, 5+).

HI-ETD-XL Cross-Linking Maps of Ubiquitin, Myoglobin, and BSA:

Inter cross-linked peptides identified by our HI-ETD-XL strategy was used to generate protein cross-linking structural maps of three model proteins Ubiquitin, Myoglobin, and BSA. The distances among the identified cross-linked lysine residues were calculated and matched with the crystal structure of bovine Ubiquitin (PDB: 1V80) illustrated in Figure S6. Inter-linked lysines identified within the ubiquitin sequence had approximate distances in the range of 20–21 Å (Table S5). A homodimer intercross-linked peptide derived from Ubiquitin protein was also identified [LIFAGK₄₈QLEDGR ………. LIFAGK₄₈QLEDGR]. Lysine 48 in Ubiquitin protein located ideally in a hydrophobic patch, reported to function as a site for chain linkage for polyubiquitination and plays a vital role in protein interactions with other substrates.[37]

Nine unique cross-linked peptides were identified from equine myoglobin and is listed in Table S7. A crystal structure of myoglobin deposited in the pdb databases was used as a model to verify the cross-linked sites and to calculate their alpha carbon distances (Cα-Cα distances) (Figure S7). We considered the spacer length of HI-ETD- XL (~14 Å), backbone flexibility of protein, and found that estimated upper limit for the Cα-Cα distances between HI-ETD-XL cross-linked lysine residues were equal to ~35 Å. Cα–Cα distances, ranging from 8.1 to 34.5 Å have reported in all the interlinked peptides identified in myoglobin. These distances matched very well within our anticipated range of cross-linking with HI-ETD-XL (~35 Å). A similar cross-linked map was generated for BSA and displayed in Figure S8.

Conclusions:

In this manuscript, we reported the development and characterization of an ETD-based cross-linker HI-ETD-XL. HI-ETD-XL cross-linked peptides preferentially cleaved by electron transfer based dissociation and showed characteristic fragmentation pattern in MS2 spectra. The signature fragmentation pattern, along with MS3 sequencing identified the cross-linked peptides quickly and unambiguously. We demonstrated the cross-linking in several model proteins and peptides. We believe this could be an effective supplemental structural tool kit for protein cross-linking and mass spectrometry studies. Another interesting fact is that hydrazone was shown to preferentially cleave by 351 nm UV laser. Fragmentations of peptides both in UVPD and ETD will also make this an efficient dual cleavable cross-linker. We believe this cross-linker can be used for benchmarking interactome and protein structural studies along with the other CID cleavable cross-linkers.

• Chemical cross-linking is a robust technique for determining protein structures

• Data complexity is one of the major challenges in protein structure analysis

• HI-ETD-XL improves overall charge density of the cross-linked peptides

• Demonstrated HI-ETD-XL cross-linking experiment with model peptides and proteins

• HI-ETD-XL will provide additional confidence in cross-linked peptide identifications along with other CID-XL cross-linkers

Significance:

Many cellular processes rely on the structural dynamics of protein complexes. The detailed knowledge of the structure and dynamics of protein complexes is crucial for understanding their biological functions and regulations. However, most of the structure of these multiprotein entities remain uncharacterized and sometimes is very challenging to reveal with biophysical techniques alone. Chemical cross-linking combined with mass spectrometry (MS) has proven to be a dependable strategy in structural proteomics field. However, data complexity and false identifications are significant hindrances for unambiguous identification of cross-linked peptides. Confident identifications demand structural studies with cross-linkers with different properties and variable spacer chain lengths. This new ETD cleavable cross-linking workflow will provide additional confidence to overcome these drawbacks and allow us to pinpoint cross-linked peptides confidently.